Rho GTPases are molecular switches that play well characterized roles in cell adhesion, polarity, and control of cytoskeletal protein dynamics in non-neuronal cells. Although functionally implicated in axon guidance and nerve regeneration, neuronal Rho GTPase cell biology is far less well understood. To address this gap, we recently used multi-mode Fluorescent Speckle Microscopy (FSM), which allows direct measurement of cytoskeletal protein dynamics, to investigate Rho dependent responses evoked by the chemorepellant agent, lysophosphatidic acid (LPA). We discovered that LPA treatment increased the contractility of "actin arc" and actin bundle structures within the growth cone and provided evidence that this contractility was driving Rho/Rho Kinase dependent growth cone retractions. Actin arc contractility appears to involve Myosin II since it depends on Myosin Light Chain Kinase and Myosin Light Chain phosphatase activities. Other work suggests Rho GTPase and Ca activity can modulate the polarity of axon guidance responses to a single ligand. Although these findings have caught the attention of axon guidance and nerve regeneration fields, the cell biological mechanisms underlying response switching are poorly understood. We have preliminary evidence that increasing background Rac activity converts LPA retraction responses to neurite growth and advance. Interestingly, such LPA evoked growth is accompanied by increases in intracellular Ca and loss of actin arc contractility -i.e. the opposite of what is observed without Rac activation. We propose to combine use of Fluorescent Speckle Microscopy and Ca Imaging to quantitatively assess actin, microtubule, and Calcium dynamics in growth cones to investigate the cytoskeletal and signaling mechanisms underlying apparent switching of LPA response polarity by Rac activity. We will also characterize the role of Myosin II in these responses and do correlative ultrastructural studies to better define the cell biology of this important molecular motor in the growth cone. Our working hypothesis is that Rac and Rho GTPases can modulate the functional output of ligand activated responses via specific effects on MT dynamics which in turn affect localization of ER Ca stores and regulate the Ca release topography and/or release sensitivity. This hypothesis will be tested in the context of LPA as well as Ephrin and Slit ligands, the latter two being known to exert their chemorepellant responses via activation of Rho in the CNS. [unreadable] [unreadable]